Project summary The mammalian brain is remarkably dynamic and can quickly adjust its functional state in response to changes in the environment. For example, when a salient event occurs, the brain enters a mode that enhances memory formation. Such brain state changes occur too rapidly to be due to anatomical rewiring. Instead, they are thought to arise from the action of neuromodulators (NMs) and neuropeptides (NPs). Unlike small-molecule NMs, such as acetylcholine and monoamines, NPs are not generally released as the major neurotransmitter from specialized neurons and they are not recycled after release. Instead most neurons synthesize and release NPs in addition to fast transmitters such as glutamate and GABA, and peptide clearance is governed by diffusion and proteolysis. Although long utilized as anatomical markers, our understanding of NP signaling is only cursory. Insights into the cellular code of peptidergic communication are only now emerging from large- scale transcriptional profiling studies that reveal the distribution of peptides and their receptors across cell types. These have revealed a differentiated anatomic distribution of NP-receptor pairs across cell types that poise NPs as important mediators of trans-cellular communication in neural circuits. However, the functional significance of NP signaling is extremely difficult, if not impossible, to study using current tools, which do not reveal the timing and location of NP signaling in vivo, or the consequences of NP signaling on neural circuit activity. Thus, new technologies are needed to enable gain- and loss-of-function studies that precisely target the normal location and timing of NP activity in behaving animals. To overcome these technical barriers, we assembled a multi-disciplinary team to develop, validate, apply, and disseminate next-generation optical toolkits for functional analysis of the spatiotemporal dynamics of NP signaling during behavior. Our toolkits include: 1) photoactivatable agents to rapidly deliver NPs (or drugs that target NP receptors) to their sites of action with high spatiotemporal precision; 2) genetically-encoded NP sensors to report when NPs are released and over what temporal and spatial scales they act: 3) new optical and genetic approaches for cell- and region-specific recording and manipulation of NP action using these probes at multiple sites in the mammalian brain simultaneously. Combining these methods with functional studies in behaving animals, we aim to establish paradigms for determining the necessity and sufficiency of NP signaling for the modulation of circuits in vivo. We will determine the context and location of NP release, the ensuing spatiotemporal pattern of NP receptor activation, and the effects this has on neuronal physiology and behavior. We will actively disseminate these toolkits to the neuroscience community. Broad applications in various brain regions and species will reveal the dynamic contribution of NPs to the control of brain circuits and plasticity. This knowledge will provide building blocks and pave the ways to refine theory and develop novel therapeutics for neurological and neuropsychiatric disorders.